There are currently no practical biomarkers for Alzheimer?s Disease (AD) that can routinely stage, assess prognosis, and monitor treatment response. AD is a complex disease of overlapping pathophysiologies that lead to eventual synapse loss and progressive dementia. The core AD biomarkers, amyloid-b and tau (total and phospho-) measured in cerebrospinal fluid (CSF), provide a strong indication of the presence or absence of substantial AD pathology, but as yet poorly reflect disease stage or progression. In order to diagnose the disease earlier, learn about the time-course of concurrent pathophysiologies, and maximize the potential for staging, tracking and treatment, it is critical that more useful AD biomarkers are characterized. A collaboration is proposed between the Yale and Massachusetts ADRCs that will leverage the individual strengths of these two centers to discover pathway driven novel biomarkers of AD. Using the large numbers of clinically characterized biofluid samples available from the Massachusetts ADRC, the Yale ADRC will employ their expertise in quantitative targeted LC- MS/MS technology to assess over one hundred analytes in CSF, with an extension of the most informative to blood. LC-MS/MS is inherently specific, and enables simultaneous testing of many markers from a small biofluid volume. Use of Data-Independent-Acquisition (DIA) LC- MS/MS techniques allows for primary data to be reanalyzed for retrospective analysis of new biomarkers, providing a reproducible yet flexible discovery pipeline. In Aim 1, a panel of over 100 proteins in CSF from pathways involved in AD pathophysiology will be subjected to comprehensive technical qualification. Using samples from the placebo arm of a clinical trial, peptides will be tested for linearity, inter-assay, intra-assay and short term biotemporal stability. All high performing peptides will then be quantified in a high contrast sample of 60 Cognitively unimpaired-adults (CU-A) and 60 dementia due to AD cases. They will be assessed for their ability to distinguish AD from CU-A, and for their correlation with markers of synaptic function. In Aim 2, technically qualified peptides will be quantified in 3 tailored sample collections to assess their performance in AD staging, prognosis and differential diagnosis. In Aim 3, the most informative analytes from Aims 1 & 2 will be quantified in blood, either by LC-MS/MS, or by sensitive or ultra-sensitive ELISA approaches. Peptide abundance will be compared across biofluids, and the diagnostic utility of these markers in blood assessed.